Circulin and process for its production



Patented Apr. 20, 1954 OFFICE CIRCULIN AND PROCESS FOR ITS PR GDUCTIGN Philip A. Tetrault, Lafayette, Ind, assignor to Purdue Research: Foundation, Lafayette, ImL,

a corporation of Indiana Application May 65.1949, Serial No. 91,857

6 Claims.

The present invention relates to a new and. useful antibiotic substance that more active against gram-negative than against gram-peel tive bacteria, herein designated. as circuli'n'.

It is an object. of the present invention to provide a new antiob'otic substance that is more active against grain-negative than against grampcsitive bacteria. A further object of the invention is to provide an organism that is capable of producing the said antibiotic substance; It is also an object of the invention to provide a process for growing or culturing the organism. G'therobjects and advantages of the invention, some of which are referred to hereinafter, will be apparent to those skilled in the art to which the invention pertains.

An organism Bacillus circulans' is described in Bergeys Manual: of. Det'erminative' Bacteriology, 6th edition, Williams and Wilkins, Baltimore, last, at page 722. 'lj'his' organism is found! in and dust. and. was first. isolated from tap water. it gives a negativeVoges-Proskauer reaction.

The particular strain. of this organism which we have isolated and which produces circulin, and we have designated as Bacillus circulcms Purdue Q 19 u. NRRL 13-1359, was isolated from thesoil. This strain gives a positive Voges-Proskauer reaction. which isat. variance with all heretofore known strains of Bacillus.- circuians.

The strain of the organism which. produces circulin, and which. we have designated Bacillus circalsms Purdue (53 1 9 u,is a gramspositivesing-le rod, measuring 0.6- to 0.8 103 2.0 tov 4.0 microns. Swollen, oval, subterminal. endspores are. present measure 3.8v to 1.0. by 1.5. to 2.0. microns.

circulates Purdue Q,v 19- 11.- produces acid, but no gas, from: the following: arabinose, dextrln, galactose, glucose,.glycerol,-insulin, lac toss, lev-ulose, 1na1tose, mannitol mannose, raffinose, rhaninosesalicin, sucrose and xylose. It reduces methylene'bluewithin.- twelve hours but becomes completely i'eoxidized after four. days. It liquefies gelatin. after five days,v digests casein and. gives a positive Voges-Proskauer reaction. It does. not utilizesodium citrate. and it does not 2, reduce nitrate. These tests were performed in accordance with the method of N. R. Smith, R. E. Gordon and 1 E. Clark, Aerobic mesophilic spore forming bacteria," U. S. Department of Agriculture, Miscellaneous Publication Number 559, Washington, 1'94'6;

The circulin-producing'Bacillus circulates Purdue. 19 u. is; noni-hemolytic' on nutrient agar containing either defibrinated rabbit or sheep blood.

The organism: can alsobe identified by and was elisecvered. by means of the zoneof inhibition it produced when it wasv grown in a. thin layer on a. nutrient-agar containing. an. appropriate dilution of. a soil suspension on top of a thick-er layer oi nutrient. agar seeded with the Lederle strain T43 of Salmonella; tylphosa.

The following table shows a comparison oi the bacterial spectra of circulin (C), polymyxin (P') and; streptomycin (8) In all these-teststhe organisms were grown for 2 l-hours at 3'7 degrees centigrade on yeast. water-veal. infusion agar, from-which they were transferred to 5 milliliters of." nutrient broth, which was then incubated for 18 to 24 hours at" 37? degrees Centigrade. One hundredthofa milliliter of this-.culturewas used to inoculate 5' milliliters of nutrient broth which contained appropriate dilutions of antibiotic. After incubation at 3.7 degrees centigrade for 24 hours. the. smallest concentration of antibiotic, inhibitory to the test organism, wasdetermined. The yeast water-veal infusion agar was prepared from milliliters yeastwater, 106* milliliters Eifco veal infusion, cue-milliliters distilled-water, 0.5 percent tryptica'se; 0 15 percent proteose-'- e tone, 0:5 percent Bacto p'eptone'; 6:5 percent sodium chloride; 0.3- percent' Bactobeef extract in 1000 milliliters of distilled water.

The antibioticsuseel for these: results assayed as follows-:-

(G). Circulin (Preparation 8835) assaying 2700 units p81- milligram.

(P) Polymyxin hydrochloride. 13-74, Batch 82,

assaying 1450 units per milligram.

(S) Streptomycin sulfate, assaying 675- units perv milligram.

BACTERIAL SPECTRA OF CIRCULIN, POLYMYXIN AND STREPTOMYCIN Inhibited by mcg. per ml. Ratios of Inhibiting oi concentrations Test Organism Strain C P S C]? (3/8 Aerobacter aeragenes NRRL Bl99 3.0 0.8 12. 5 4 0.25 Bacillus anthracis K 100.0 100. 0. 8 1, 030 B. biilz' K 100. 0 100. 0 12. 0 Brucella bronchzsep NRRL B139 0. 8 0. 1 100. 0 0. 01 Escherichia coli AT 0 0. 8 0.8 25. 0 1 0.03 E. cali NRRL 13210..-. 6.0 6.0 25. 0 0. 2 Klebsz'ella prim-maniac 0. 8 0. 4 3.0 O. 2 K. pneumoniae 13 0. 4 0. 4 3. 0

0.02 0.02 3 0 1 0.007 Do Be PR 150.0 75.0 0 2 6 0 Micrococcus pyogenes var.

aureus 100. 0 100. 0 100. 0 M. pyogenes var. aZbus.. 25. 0 100. O 3. 0 Neisserz'a catarrhalis K 3.0 6.0 3. 0 Pseudomonas aerugz'nosa 6. 0 6.0 50. 0 Ps. aeruginosa 3. 0 18.0 50. 0 D 3. 0 3.0 100. 0 0. 8 0. 8 12.5

1. 6 0. 2 25. 0 8 0. 06 3. 0 1. 6 25. 0 2 0. 12 S. gallinarum 0. 4 0. 2 50. 0 2 0. 08 S. paratyphi. 6. 3 l. 6 50. 0 4 0. 12 Do 1. 6 0. 8 25. 0 2 0. 06 S. pullorum 0. 8 0. l 50. 0 2 0. 02 S. paratyphi 1. 6 l. 6 100. 0 1 0. 01 S. schnttmuellerz'. 6. 0 0.8 50. 0 8 0.12

0. 1 0. 1 50. 0. 02 D 0. 4 0. 4 100.0 Do... 0. 4 0. 4 100. 0

Do 3. 0 0. 4 100. 0 Shigella dysenterzae K 0. 4 0- 4 1. 6

ISOLATION OF BAC'ILLUS CIRCULANS PURDUE Q 19 11 CULTIVATION OF BACILLUS CIRCULANS Purdue Q 19 u Although the organism can be cultivated on nutrient agar slants or plates, seed cultures suitable for inoculation of carbohydrate-containing nutrient media suitable for and promoting the production of circulin are best prepared from media described and claimed in the copending application of Donald R. Colingsworth, Serial 91,859, filed concurrently herewith; such a medium is one typified by that having the following composition:

Rolled oats grams 2.0 Dextrin do 2.0 Ammonium sulfate (NH4)2SO4 do 1.0 Potassium chloride KCl do 0.4 Calcium carbonate CaCos do 1.0 Tap water, q. s mi1liliters 100 The sterile medium can be inoculated with a culture from an agar slant and the organism grown at an optimum temperature of approximately 2% degrees centigrade under aerobic submerged conditions. Specific methods of cultivating the organism, which are described in the said copending application of Donald R. Colingsworth, are included in the examples herein.

The circulin can best be isolated from the fermentation broth by procedures described in the copendlng application of Durey H. Peterson and Byron E. Leach, Serial 91,858, filed concurrently herewith. Certain of these procedures are described in the examples hereinafter. The process produces two circulin fractions, A and B, of which circulin A is the predominating fraction. DOWling et al., Science 116, 147 (1952); Peterson and Reineke, J. Biol. Chem., 181, (1949).

PROPERTIES OF CIRCULIN Circulin is a polypeptide containing free amino groups and amide (peptide) linked nitrogen. Although pure circulin salts are remarkably stable, the free base, obtained at a pH of approximately 11.0, is unstable and is best recovered, as is streptomycin, in the form of its salts, such as the sulfate, hydrochloride and others, the activity of these salts in adversely modifying the vital functions of specific microorganisms being pro-' portional to their circulin content. Pure circulin has an activity of approximately 6300 E. soli units per milligram, assayed in accordance with Murray et al., J. Bact. 57, 305 (1949).

Circulin sulfate is an amorphous solid having a decomposition point of approximately 226 to 228 degrees centigrade, [a] =minus 61.6 degrees (c.=l.25 in water). It gives a negative Sakaguchi test, indicating the absence of the amino acid arginine; a negative xanthoproteic reaction, indicating the absence of aromatic amino acids; negative Knoops test for histidine and contains no carbon-sulfur linkages. Nitrous acid (Van Slyke reaction) produced deamination and reduces the activity to approximate 5 percent that of the original circulin. Analysis of circulin sulfate shows: C, 44.60 percent; N (Dumas), 16.0 percent; H, 7.47 percent; and ionic sulfate, 21.3 percent. Circulin sulfate has an approximate molecular weight of 1200 based on its ionic sulfate content and an empirical formula of CssiI-I7iO9N122 H2SO4 or a multiple thereof. Approximately five of the nitrogens are present as amino nitrogen, the remainder being in amide linkage, as is shown by analysis for amino nitrogen before hydrolysis, which is 57.5 percent, and amino nitrogen analysis after hydrolysis, which is 15.8 percent with no free ammonia being evolved. Circulin sulfate when digested with a 1 percent crnde'trypsin solution containing lipase at 37 degrees centigrade and 850 to 8.'5 shows a loss of antibiotic activity against E. coli of from 50 to 70 percent in 'from 96'to '144hours. Analysis for amino acids on acid-hydrolyzed ciroulin by the, paper chromatography procedure of Consden, Gordon Martin, Biochem. ,J., 1944, vol. 38, page 224, shows the presence of -d-leucine -('7 to 8 percent), d-threonine (8 to 9 percent), and

alphagamma-diaminobutyric acid and the absence of serine and phenylalanine. Ether extraction of the acid-hydrolyzed material gives pelargonic or an optically-active acid isomeric with pelargonic acid. Circulin sulfate has no significant peaks in its ultravioletabsorption spectrum, which is indicative of the absence of phenyl groups.

Circulin hydrochloride has :a melting point of 232 to 236 degrees centigrade with decomposition, [al =minus 60.;1 degrees -(c.=2.094 in water), and assays approximately 6500 units per milligram. Circulin hydrochloride in a mineral oil mull exhibits characteristic absorption in the infrared region :of the spectrum at the following wave lengths in microns: 2.9, 3.1, 4.4, 5, 5.6, 6, 6.3, 6.6, 7.2, 7.3., 7.6, 18218.5, 8.7, 9.8., 10.1, 10.8, 11.3, 11.9, 12.9, 13.2, 14a-nd 15 as shown in Figure 1 of the accompanying drawing. The picrate assays 3200 to 3600 units 'per milligram and has a melting point of 160 to 168 degrees centigrade with decomposition. The :helianthate assays 2600 to 328% units per milligram and has a melting point of 218 to 222 degrees centigrade with decomposition. The .re'ineckate assays 2400 to 3300 units per milligram rand :darkens at :a tempera ture of 185 to 19.5 degrees centigrade but undergoes no further visible change upon heating to 2&0 degrees centigrade.

Circulin is insome respectssimilar to the antibiotics designated in the art and herein as polymyxin A, polymyxin 'B, polymyxin C and polyinyxin D. Polymyxin A, B and C are also .lmown as aerosporins A, B and C, while polymyxin D is also called polymyxin without a distinguishing letter. Circulin differs from 'polymyxins B, C and D in chemical composition as is shown in the following tableof amino acid components:

Polymyxin (polymyxin D) is not affected by digestion with pepsin, pancreatin, erepsin and trypsin (P. G. Stansly and N. H. Ananenko, Archiv. Biochem., 1947, vol. 15, pages 473-5.) and crude trypsin does not affect ,polymyxins A and C, but circulin and polymyxin B are destroyed to the extent of 50 to 70 percent in "96 to 144 hours by digestionwith crude 'trypsin, yet neither circulin nor polymyxi-n 1B are inactivated by crystalline trypsin. Polymyxi-n Band circulin differ in theiramino acidcontents. Though polym'yxin A and circulin have :similar amino :acid content they differ markedly in their reaction toward lipase.

The antibiotic substance *of this invention is prepared by cultivating 1a circulin-producing strain of Bacillus circuluns, .such as Bacillus .cz'r- .culans Purdue Q .19 'u, preferably under submerged aerobic conditions, in a nutrient medium containing a carbohydrate, an ammonium salt, potassium dihydrogen phosphate, and an inorganic buffer system. vA preferred medium :comprises dextrin together with dried 'ibrewerls yeast or rolled oats as .a source of protein and to stimulate circulin production. The circulin thus produced is separated from the culture medium, :aft'er destruction of the organism and coagulation "of the protein material by heat, either by precipitation as a complex with alky-lated aromatic sulfonates, or by adsorption on activated charcoal, eluting the antibiotic from the adsorbate with acidified aqueous alcohols having from 3 to 5 carbon atoms, inclusive, recovering the crude circulin from the eluate and purifying the crude product by chromatographic absorption of aqueous solutions on activated :carbon.

For the preparation of circul-in, a culture medium which is preferred is one containing approximately:

Parts Dextrin or starch 20 Ammonium sulfate (NH4D2SO4 10 Dried brewers yeast or rolled :oats 20 Potassium chloride (KCl) 4 Calcium carbonate (CaCOs 10130 16 Potassium dihydrogen phosphate (KI-12 304) o 012 1:00.4 Water, qns 1000 The medium is inoculated with a vegetative culture (as opposed to aspore culture) "of ,a :circulin-producing strain of Bacillus circulanapreferably in an amount :of about 5 percent by volume of the nutrient medium. The inoculating culture is obtained :by transferring the circulinproducing strain of Bacillus 'cz'rculans grown on agar slants to loo-milliliter portions of the corn sugar-brewers yeast medium having the composition specifled hereinabove but containing no dihydrogen potassium phosphate .and wherein the dextrin can .be replaced .by acorn sugar, and incubating it under submerged aerobic conditions at 24 degrees centigrade until the .fi-rst signs of spore formation appear. This .inocu'lum may be used directly in large-scale :Eermentations but is preferably transferred to 5-gallon bottles containing 3 liters of the corn sugar-brewers yeast medium specified hereinabove containing dihydrogen potassium phosphate. The culture of Bacillus .circu'lans thus obtained -contains vegetative growth, which is the preferred :form for inoculation, and may be added to large-scale formenters in an amount equal to approximately '5 percent by volume of the medium used.

The fermentation is conducted at 24 degrees centigrade for from two to five days with the maximum yield being obtained shortly after 48 hours of incubation. vAir is passed into the agitated, inoculated medium at a preferred .rate of about 0.6 volume of air per volume of medium per minute. Iron should be. avoided as they retard the formation of .circulin, although iron does not itself prevent growth of the organism.

After the fermentation proceeded for the desired length of time, :as determined cbyiassay of the brew for circulin content, the brew is heated in the fermenter to 100 degrees centigrade for about minutes to kill the Bacillus circulans, open the bacterial cells, coagulate the suspended material and otherwise aid in the subsequent filtration. The hot brew is cooled to between approximately 25 and approximately 60 degrees Centigrade and filtered, usin a filter aid. The brew may be acidified to a pH within the range of 2 to 3 with sulfuric acid prior to filtration, if desired. The circulin is recovered from the clarified beer by adsorption on activated charcoal or i precipitated therefrom by the addition of certain surface-active agents such as alkylated aromatic sulfonates, oleic acid esters of sulfonated aliphatic compounds and aliphatic ester sulfates, which form complexes therewith. A diatomaceous earth filter aid is preferably added to assist in the separation by filtration of the circulin from the brew.

The circulin-containing carbon filter cake is eluted several times with an aqueous solvent, preferably 50 percent aqueous tertiary-butanol acidified to a pH within the range of 2 to 4 with sulfuric acid. Other concentrations of tertiarybutanol are effective as are aqueous propanol, isopropanol, other isomeric butanols, pentanols and acetone. Ethanol is not satisfactory as an eluting agent nor are the hexanols of much practical value.

The eluates are concentrated to about 10 percent of their original volume, the eluting organic solvent being removed during the concentration. lhe excess sulfuric acid in the eluting solvent is neutralized by sodium or barium hydroxide,

barium hydroxide .beingpreferred as the excess sulfate ion precipitates barium sulfate which can be removed from the brew by filtration. The resulting solution may be used directly for further purification but is preferably frozen and dri d, the resulting solid being then redissolved for further purification.

The crude circulin as obtained can be converted to pure material by chromatography using activated carbon as the adsorbent and neutral or aqueous tertiary-butanol as the developsolvent. There is thus obtained purified circulin in a form suitable for therapeutic use.

Although circulin of satisfactory purity can be obtained by adsorption on carbon and elution with solvents containing acid, products of greater purity and potency can be more readily obtained and the overall yield of the process can be increased by extraction of the circulin as a complex from the fermentation brew with water-immiscible organic solvents. In practicing such alternative processes, a sulfonated or sulfated surfaceactive agent is preferably added to the clarified or filtered brew, whereby the circulin complex precipitates. This precipitate can be separated from te brew and the circulin recovered therefrom. some surface-active agents may not form compl xes with circulin that are water-insoluble, but may nonetheless aid in the partition of the circulin between the aqueous and the Waterimmiscible solvent phases.

Sulfonated compounds which are surface-active agents and which form a complex with circulin are:

l. Dodecylbenzene sulfonic and similar alkylated aromatic sulfonic acids and Water-solule salts thereof, such as are available under the trade-names Santomerse D, Naccanol NR, Merpentine, Ahcowet, Aresklene, and Ultrawet 3013s.

2. Salts of sulfosuccinic and similar acids, such cm (CH2) 7CH=CH (CH2) 7-COO-C2H4-SO3Na which is sold under the trade-name Artic Syntex A.

4. Sodium salts of aryl alkyl ether sulfates such as those available under the trade-name TritonJ r 5. Sulfonated naphthalene alkyl ethers such as are available under the trade-name Tenisol.

6. Aliphatic ester sulfates.

The precipitated complex of circulin and the sulfonated or sulfated surface-active agent can be separated from the fermentation brew by filtration (using Celite or other filter aids, if necessary) or centrifugation, and thereafter the circulin can be recovered therefrom by dissolving the precipitate in an organic solvent such as normal-butanol, amyl acetate or other suitable solvent such as methanol and chloroform, concentrating the solution by evaporation, preferably at subatmospheric pressure, followed by treatment of the solution with hydrogen chloride or concentrated hydrochloric acid. Alternatively, concentrated sulfuric acid may be added to form circulin sulfate at this point. In such treatment,

acetone may be added to the normal-butanol to aid in the further precipitation of circulin hydrcchloride.

Alternatively, the fermentation brew containing the circulin and surface-active agent, whether or not a complex is formed or precipitated therein, may be treated directly with normalbutanol, amyl acetate, chloroform or other waterimmiscible solvent, whereby circulin is extracted therefrom.

The following examples are illustrative of the invention but are not to be construed as limiting.

EXAMPLE 1.PREPARATION OF SEED CUL- TURES OF BACZLLUS CIRCULANS PURDUE Q 19 u The vegetative growth and spores of Bacillus ci'rculans Purdue Q 19 u, grown on agar slants, was transferred to several SOD-milliliter flasks, each containing milliliters of the following medium:

Rolled oats grams 2.0 Dextrin do 2.0 Ammonium sulfate (NH4 2SO4 do 10 Potassium chloride KCl do 0.4 Calcium carbonate CaCO3 do 1.0 Tap water, q. s. milliliters 100 This medium was sterilized and inoculated from agar slants. It was shaken (aerated, submerged culture) for 48 hours at a temperature of 24 degrees centigrade. This culture of Bacillus circalans material can be used directly for the inoculation of circulin-producing tanks (Example 3) or for the production of a larger volume of inoculum (Example 2).

EXAMPLE 2.--PREPARATION OF INOCULUM To six liters of a medium containing:

Corn sugar grams 20 Dried brewers yeast do 20 Ammonium sulfate (NH4)2SO4 do 10 Potassium. chloride KCl do 4.0

9 Calcium carbonate CaCOB; grams 16.0 Potassium dihydrogen phosphate KH2PO4 do 0.2 Water, q. s liter 1 in a 5-gallon flask, equipped for agitation and aeration, was added five percent by volume of a EXAMPLE 3.-PREPARATION OF CIRCULIN SULFATE A. Fermentation Two one-hundred-gallon fermentation tanks, each containing 240 liters of a medium containing per liter:

Dextrin or starch grams 20 Ammonium sulfate (NH4)2SO4 do Dried brewers yeast do 20 Potassium chloride KCl do 4.0 Calcium carbonate CaCOs do 16.0 Potassium dihydrogen phosphate KH2PO4 dO 0.2 Water, q. s liter 1 were inoculated with 400 milliliters each of shake flask cultures of Bacillus circulans Purdue Q 19 u obtained as described in Example 1. The inoculated medium was incubated for 88 hours at a temperature of 24 degrees centigrade with aeration and stirring. Assays of portions of the medium during fermentation shows the following quantities of circulin per milliliter of brew in the respective tanks:

Run A-71, Run A-72 Units Units B. Isolation At the end of the fermentationperiod the contents of tanks A-71 and A-72 above were heated to 100 degrees centigrade for 10 minutes, which killed the organism, openedthe bacterial cells and coagulated suspended material. The pH of each tank was adjusted to about 2.0 with concentrated sulfuric acid, about 2100 milliliters being required for each tank. The contents of each tank were then allowed to cool to about 25 degrees centigrade and to each was added with stirring, 10.6 kilograms (about 5 per cent weight/ volume) of a diatomaceous earth filter aid (Johns-Manville Celite-545), the suspension filtered. and each filter cake washed with one-tenth volume (21 liters) of water.

The filtrates and washes were combined, the pH adjusted to 6.5 with sodium hydroxide, 1 kilograms of filter aid (Supercel) added and the suspension was filtered. A portion of the filtrate was withdrawn for other purposes, leaving 355 liters of clarified brew which contained, as shown by assay, 412 million units of circulin. I

The circulin was adsorbed from the brew by the addition of 315 kilograms of activated carbon (Darco G 60) and stirred for forty-five minutes.

circulin per milligram.

Eight hundred grams'of a diatomaceous earth filter aid (Cents-545) was added, the suspension filtered and the filter cake washed free of sulfate ion with water and then liters of 50 percent aqueous tertiary-butanol. The tertiary-butanol wash, containing 30.3 million units of circulin, was discarded.

The circulin adsorbed on the filter cake (contained on a. plate and frame filter press) was eluted by circulating solvent through the filter press until equilibrium was reached and then withdrawing the eiuting solvent. This elution was conducted stepwise, as follows:

ELUATE No. 1

Ten liters of water containing 500 milliliters of tertiary-butanol was circulated through the filter press, the tertiary-butanol remaining on the cake from the previous wash being sufficient to raise the tertiary-butanol content of the eluting solvent to 10 percent. Concentrated sulfuric acid was added to the circulating solvent until the pH remained constant at about pl-l 3.85. The eluting solvent, which was then withdrawn from the filter press, amounted to 10.5 liters and contained 42.5 million units of circulin. Assay of an aliquot portion showed the solids contained 530 units of circulin per milligram.

ELUATE NO. 2

The filter cake remaining on the press was eluted by circulating 10 liters of 50 percent aqueous tertiary-butanol acidified to pH 4.0 with sulfuric acid through the press. When the eluting solvent was withdrawn a volume of 9 liters was obtained which contained 46.5 million units of circulin. Assay of an aliquot portion shows that the solids contained 1190 units of circulin per milligram.

ELUATE NO. 3

The filter cake was eluted by circulating 10 liters of 50 percent tertiary-butanol acidified to pH 4.0 with sulfuric acid. When the eluting solvent was withdrawn there was obtained a volume of 8.5 liters containing 21.2 million units of circulin, the solids containing 1000 units of The filter cake was eluted by circulating through the press 10 liters of.50lpercent tertiarybutanol acidified to pH 2.8 withsulfuric acid. A

volume of 9.75 liters of eluate was withdrawn which contained 19.8 million units of circulin, the solids assaying 695 units of circulin per milligram.

ELUATE NO. 5 I

The filter cake was eluted as above with 10 liters of 50 percent tertiary-butanol acidified to pH 2.2 with concentrated sulfuric acid. There was collected 9.5 liters of solvent containing 13.3 million units of circulin; the solids assaying 480 units of circulin per milligram.

The five eluates were combined and concentrated under reduced pressure to a volume of 4.5 liters. Barium hydroxide solution was added until the solution had a pH of 6.0, the excess sulfate ion not combined with circulin being removed by filtration as barium sulfate (sulfate ion attached to circulin is removed at pH values of about 11.0). The suspension was filtered to remove the precipitated barium sulfate and the filtratewasfrozen and dried. .There was obtained 147.0 grams-of. crude circulin sulfate as.-' saying. 880 units .ofcirculin .permilligram, a re- 'covery of 34 percent. ofthe total activity.

ll C. Purification,

1. PREPARATION OF AN ACTIVATED 'CARBON COLUMN One thousand grams of an acid washed, heat-- activiated carbon (Darco G 60'), 1000 grams of a diatomaceous earth filter aid (Celit-el-5') and 6 liters of water were thoroughly mixed. Sulfuric acid was added during the mixing until the pH of the supernatant liquid remained constant at a pH of 3.93. The suspension was filtered, the filter cake resuspended in 3.0 liters of water acidified to pH 4.0 with sulfuric acid, filtered, and the filter cake washed twice with one-liter portions of water acidified to pH 4.0 with sulfuric acid. The carbon-diatomaceousearth mixture was suspended in a small amount of aqueous sulfuric acid .(pH 4.0) and placed in an upright glass column, 10 x 125 centimeters, equipped with a retaining plate and stopcock for liquid withdrawal at the bottom and a closed'top with stopcocks adapted for the addition of liquids and air under a pressure of 10 to .25 pounds per square inch. The carbon-diatomaceous earth mixture cc- .cupied a height in the column of 62.5 centimeters and had a liquid holdup of 4.5 liters.

2. CHROMATOGRAPHY 0F amour-11v SULFATE The 147 grams of crude circulin sulfate obtained in Example 33 was dissolved in 520 milliliters of dilute sulfuric acid having a pH of 4.3 and placed on the activated carbon column above. The outlet was adjusted to deliver 0.5 milliliter per square centimeter per minute, a total fiow of 39.8 milliliters per minute, under a pressure of about pounds per square inch.

The 520 milliliters of circulin sulfate solution was followed by 5000 milliliters of dilute sulfuric acid (pH 4.0) and then aqueous '25 percent tertiary-butanol acidified to pH 4.0 with sulfuric acid. The solutions after passing through the column were withdrawn at the bottom, pooled, the solvent removed and the dried solids thus obtained assayed for circulin. The results obtained were as follows:

. Activity Grams of Volume of Fraction Milhhters) per 501m Milligram 28. 14 0 T. 62 185 5. .60 L 4.65 29. 4 3, 300 0. 3 '1, 075 l. 87 .575

The material having an activity of 3300 units per milligram, after .a second chromatographic absorption, yielded circulin sulfatehaving an activity of approximately 5000 to 6000 units per milligram whose activity was not increased by further treatment. 7

EXAMPLE 4.-PREPARATION AND PURIFI- CATION OF CIRCULIN HYDROCE'LORIDE A. Fermentation Two one-hundred-gallon fermentation tanks, each containing 240 liters of a medium having the following composition:

1 Bonn-119., Run A120,

Units Units B. Isolation At the end of the 72-houriermentation period the contents of the two tanks were heated to degrees centigrade for 10 minutes, which killed the organism, opened the bacterial cells and coagulated suspended material. The contents "of each tank were cooled to about 60 degrees centigrade and to each was added, with stirring, about kilograms ofa diatomaceous earth filter aid iJohns-Manville Cents-545) and the suspension was filtered. There was obtained .a total of 400 liters of filtered brew 3.385 million units of circulin.

One liter of Ultrawet 30DS (solution of mixed sodium salts of alkylated aromatic sulfonic acids containing approximately 25 percent active sulfonate), 4.0 kilograms of activated carbon (Darco G 60) and 4.0 kilograms of a diatomaceous earth filter aid (CBIitBufifl) were added, with stirring, to 200 liters of thefiltered brew and the resulting suspension filtered on a plate and frame filter press.

The filter cake was eluted once with .20 liters of tertiary-butanol acidified to pH 1.7 with su furic acid and three additional times wit 22- liter portions of 50 percent tertiary-butanol, acidified to pH 1.9 with sulfuric acid. The eluates were combined, the pH adjusted to 5.0 with milliliters of saturated sodium hydroxide solution and concentrated toa volume of 4.5 liters under a pressure of 4 inches of mercury and at a temperature of 45 degrees centigrade.

To the concentrated eluates was added a solution of .500 grams of picric acid dissolved in one liter of acetone. The acetone was removed at subatmospheric pressure and the circulin picrate, which had separated, was collected and'washed with 5 liters of water. The circulin picrate was dissolved in 15 liters of acetone, the solution filtered, and concentrated hydrochloric acid (or dry hydrogen chloride) was added to the filtered acetone solution until the precipitation oi circulin hydrochloride was complete, 55 milliliters of concentrated hydrochloric acid'being required. The precipitated circulin hydrochloride was collected, washed with 3 liters of acetone and then dissolved in 2.5 liters of water. The pH of the solution was adjusted to 6.8 with saturated sodium hydroxide solution and filtered and the filtrate was subsequently frozen and dried. There was obtained 106 grams of crude circulin hydrochloride which assayed 2015 units of circulin per milligram, a recovery of 23 percent of the total activity.

C. Purification of circulz'n hydrochloride A mixture of 1200 grams ofactivated carbon (Darco G 60), 1200 grams of a diatomaceous earth (Celite) and 700 milliliters of acidified distilled water having a pH of 4.75 were placed in the glass column described in Example 3C. The solids occupied a height in the column of 62.5 centimeters and had a liquid holdup of 5000 milliliters.

A solution of 148.0 grams of the circulin hydrochloride above (activity 2015 units per milligram) in 275 milliliters of 10 percent aqueous tertiarybutanol, which now assayed 1720 units per milligram or a total of 255 million units, was added to the column, washed down and developed with aqueous 10 percent tertiary-butanol under a pressure of 10 pounds per square inch. The solutions after passing through the column were withdrawn at the bottom, pooled, the solvent removed and the dried solids thus obtained assayed for circulin. The results obtained were as follows:

There was thus obtained 25.14 grams of circulin hydrochloride having an activity of 5700 to 6300 units per milligram. Further chromatography of this material has not increased the units of activity per milligram.

Example A fermenter containing 240 liters of the medium described in Example 4 was inoculated and incubated as described in Example 4, assays 01 the brew for circulin showing the following quantities of circulin per milliliter of brew: 48 hours, 2280 units; 64 hours, 3680 units.

At the end of the fermentation period the contents of the fermenter were heated to 100 degrees centigrade for ten minutes, cooled to 60 degrees centigrade, kilograms of a diatomaceous filter aid (Celite-545) added, the suspension filtered and the filter cake washed with water. The filtrate and washings were combined, one liter of Ultrawet 30DS (as in Example 4), 4 kilograms of activated carbon (Darco G 60) 2 kilograms of a diatomaceous earth filter aid were added and the suspension stirred for thirty minutes filtered in a plate and frame filter press. The filter cake was eluted three times with respectively 22-, and 15-liter portions of 80 percent acetone acidified to pH 2.0 with sulfuric acid and then with 15 liters of water. The acetone eluates were combined and then divided into two equal portions for further treatment. The water wash (13.5 liters) was further treated by adding sixty-seven and one-half liters of acetone. The crude circulin sulfate which. precipitated was collected, dissolved in water, the solution filtered, the pH of the filtrate adjusted to pl-l 5.5 with sodium hydroxide solution, filtered again, and the filtrate was frozen and dried. There was obtained 37.5 grams of solid assaying 650 units of circulin per milligram.

One-half of the acetone eluates (24.75 liters) containing 77.2 million units of circulin was neutralized to pH 6.5 and concentrated to a volume of 4.0 liters under sub-atmospheric pressure. The concentrated, acetone-free solution was extracted twice with 4-liter portions of dilute sulfuric acid having a pH of 1.2. The butanol which had dissolved in the water 'was removed b distillation at sub-atmospheric pressure, the resulting aqueous solution, amounting to 4.75 liters, was neutralized to pH 5.0 with sodium hydroxide solution, and wa subsequently frozen and dried. There was obtained grams of solid assaying 170 units of circulin per milligram.

To the second half of the acetone eluate (24.75 liters) was added 99 liters of acetone, the circu lin sulfate which precipitated was collected, dissolved in water and the cloudy solution filtered. The pH of the filtrate was adjusted to 5.5 with sodium hydroxide, and it was filtered to remove a small amount of precipitate, and was thereafter frozen and dried. There was obtained 36.5 grams of solid assaying 1440 units of circulin per milligram.

EXAMPLE 6 A fermenter containing 215 liters of the medium described in Example 4 was inoculated and incubated as described in Example 4. Assays of the brew for circulin showed 4080 units per milliliter or a total of 880 million units after 64 hours.

At the end of the fermentation period the contents of the iermenter were heated to 100 degrees centigrade for ten minutes, cooled to 35 degrees centigrade, 10 kilograms of diatomaceous earth filter aid (Celite-545) added, the suspension filtered and the cake washed with sufficient water to bring the volume of the filtrate to 215 liters.

One and seven-tenths liters of Ultrawet BODS (as in Example 4) were added and the suspension was stirred for ten minutes; 2150 grams of di atornaceous earth filter aid (Supercel) were then added, the suspension stirred for ten minutes and an additional 4300 grams of diatomaceous earth filter aid was added and the suspension stirred for ten minutes, and the whole was filtered in a plate and frame filter press. The filter cake was eluted three times with respectively 80-, 20- and 20-liter portions of 80 percent acetone acidified to a pH between 1.5 and 1.35 with sulfuric acid. The Volume of the pooled eluates was 62.0 liters and its circulin content was 658 million units.

The residual cake was washed with 31 liters of water acidified to pH 1.6 with sulfuric acid, the eluate containing 71 million units of circulin.

To the 62 liters of 80 percent acetone eluate above, 248 liters of acetone and grams of diatomaceous earth (Supercel) were added and the suspension was filtered. The filter cake was suspended in 1750 milliliters of water, which dissolved the circulin, and filtered. The pH of the filtrate was adjusted to 5.2 with 14 milliliters of saturated sodium hydroxide solution, filtered, and the residue washed with 4.00 milliliters of water. The filtrates and washings were combined, frozen and dried from the frozen state and 143.5 grams of circulin having a potency of 4640 units per milligram was obtained.

To the 31 liters of water wash, following the acetone elution above, wa added 124 liters of acetone and 100 grams of diatomaceous earth (Supercel) and the suspension filtered. The filter cake was suspended in one liter of water, the suspension filtered and the residue washed with 100 milliliters of Water. The pH of the filtrate was adjusted to 5.7 with sodium hydroxide solution, 50 grams of diatomaceous earth filter aid (Supercel) was added, the suspension filtered and the filtrate was frozen and dried. There was terations may-be made therein in conventional,

manner in. accordance with the principles herein described, and that the invention is not to be limited except as definedin. the appended claims.

I claim: I 1. The substance designated circulin, which (a) is'efiective in inhibiting. the growth of gramnegative bacteria; (12) is a; basic polypeptide containing .ieucine; threonine, alpha, gamma-diaminobutyric;acidan'dan optically active isomer of pelargonic acid; contains free amino groups, as determinedby theivan Slylze reaction; (d) forms salts with .'acids;. (c) has an amino nitrogen analysis of "7.5 percent before hydrolysis and-B percent afterhydrolysis; (7) is substantially destroyed by crude trypsin and lipase; forms a sulfatethat is anamorphous solid soluble in water. and to a lesser degree soluble in lower alcohols but insoluble. in acetone and water-immiscible solvents. the sulfate salt having a melting point of. 226 to 228 degrees centigrade, a specific rotation [001 of minus 61.6 degrees (c.='1.25 in water) and (h) forms a hydrochloride salt exhibiting in a m neral oil suspension a characteristic absorption. in the infrared region of the spectrum at the following Wave lengths in microns: 2.9, 3.1, 4.4, 5, 5.6, 6, 6.3, 6.6,

16" 7.2, 7.3, 7.6 ,i-8.2,.8.5, 8.7,:9.8, 10.1, 10.8, 11.3, 11.9, 12.9, 13.2,? 14;:and 15*as1shown in Figure 1 oi -the accompanying drawing.

2. A product ofthe group consisting of circulin as defined iniclaiinl and acid salts thereoi.

3. A process for the production of the substance circulin as described in claim 1 which comprises aerobically-cultivating a substantially pure culture of the Purdue Q 19 11 strain ofthe organism Bacillus circulans. upon acarbohydrate-containingnutrient'medium andrecovering the circulin therefrom.

. 4. A process for the production of the substance circulin as defined: in /claim 1- which comprises cultivating asubstantially pure culture of the Purdue Q 19 u strain of the organism Bacillus circulcms uponan aqueous carbohydrate nutrient mediumand recovering the eirculin therefrom.

5.. Circulinsulfate, the sulfate of the substance defined in claim 1.

6. Cir'culin hydrochloride, the hydrochloride of the substance defined in claim 1.

.itcierences Cited insthe. file of this patent Shepherd, on'Polymyxin, in J. A. C. 6., volume 70, November. 1948, pages 3771 to 377%.

Stansly, on Polymyxin, in Bull. Johns Hopkins Hospital, volume 81,-September 11, 1947, pages 43 to 54.

.Garson' et al., indournal Bacteriology, volume 58, July 19.49, in Notes-pages a'nd 116.

McLeod, .on Circulin, in Journal Bacteriology, volume 56, December 1948, pages 749 to 754.

Bowling et al.: Science, volume 116, Number 3066, August 8, 1952; pages 147 and 148.

Murray et a1.: Journal Bacteriology, volume 57, pages 305 to 307, March 194:9.

i/anderbrook et al.i J. Clin. Invt, September 1949, pages 1032 t0'1035.

Bergey: Manual of 'Determinative Bacteriolo y, 6th edition, 1948; pages 722, 723. 

1. THE SUBSTANCE DESIGNATED CIRCULIN, WHICH (A) IS EFFECTIVE IN INHIBITING THE GROWTH OF GRAMNEGATIVE BACTERIA; (B) IS A BASIC POLYPEPTIDE CONTAINING LEUCINE,THREONINE, ALPHA, GAMMA-DIAMINOBUTYRIC ACID AND AN OPTICALLY ACTIVE ISOMER OF PELARGONIC ACID; (C) CONTAINS FREE AMINO GROUPS, AS DETERMINED BY THE VAN SLYKE REACTION; (D) FORMS SALTS WITH ACIDS; (E) HAS AN AMINO NITORGEN ANALYSIS OF 7.5 PERCENT BEFORE HYDROLYSIS AND 15.8 PERCENT AFTER HYDROLYSIS; (F) IS SUBSTANTIALLY DESTROYED BY CRUDE TRYPSIN AND LIPASE; (G) FORMS A SULFATE THAT IS AN AMORPHOUS SOLID SOLUBLE IN WATER AND TO A LESSER DEGREE SOLUBLE IN LOWER 